真菌生物碱Brevianamides的生物合成机制与合成生物学研究

Brevianamides are important fungal alkaloid insecticides. It has been long hypothesized that the brevianamide biosynthetic pathway might contain a unique Diels-Alderase responsible for formation of the bicyclo[2.2.2]-diazaoctane moiety in brevianamide structures. To test this "Holy Grail" hypothesis in the field of biosynthesis, starting from genome mining of the brevianamide biosynthetic gene cluster, we will stepwise reconstitute the entire brevianamide biosynthetic pathway and study the related catalytic mechanisms. Moreover, this project is expected to generate novel brevianamide derivatives with a wider-spectrum of bioactivities by taking advantage of synthetic biology methods including heterologous expression, selective assembly of gene cluster, pathway reprogramming via introduction of heterologous tailoring enzymes, and substrate analougue feeding. These novel brevianamide compounds could become candidates for future drug discovery.

Brevianamides是真菌生物碱类杀虫抗生素，其生物合成途径中可能含有一种稀有的D-A酶，通过催化[4+2]分子内Diels-Alder反应形成独特的双环[2.2.2]-重氮辛烷结构单元。为了验证这一生物合成领域的重大假说、捕获D-A酶，本项目一方面将从brevianamide生物合成基因簇的挖掘出发，在体外分步重建整条brevianamide生物合成途径，阐明催化机制；另一方面将通过基因簇异源表达、选择性组装以及基于异源修饰酶和底物类似物的化学结构定向改造等合成生物学方法产生具有新结构和新活性的brevianamide结构衍生物，为未来的新药开发提供候选化合物。

Brevianamides是真菌生物碱类杀虫抗生素，其生物合成是通过[4+2]分子内Diels-Alder反应形成独特的双环[2.2.2]-重氮辛烷结构单元。本项目从brevianamide生物合成基因簇的挖掘出发，利用基因敲除、异源表达和体外酶反应重建等方法解析了brevianamide生物合成途径，并阐明了相关生物合成酶的催化机制。我们不仅完成了brevianamide A的异源宿主全合成，而且发现和鉴定了一个催化机制独特的异构酶，为最终确定Diels-Alderase奠定了重要基础。在生物合成基因异源表达过程中，通过不同酶的组合生成了一系列具有新结构的brevianamide结构衍生物，将为未来的新药研发提供候选化合物。